Regulation of RNA condensates by RNA helicases in development

PROJECT ABSTRACT RNA condensates, also known as membraneless organelles or RNA granules, are dynamic concentrations of protein and RNA that spontaneously coalesce in the absence of a limiting membrane within the cytoplasm or nucleoplasm through liquid-liquid phase separation. Eukaryotic cells harbor various RNA condensates, including nucleoli, processing bodies, stress granules, and germ granules. Functioning as distinct states of liquid, these droplets and their surrounding cytoplasm play a crucial role in concentrating reactants for cellular organization and facilitating specific biochemical reactions during development. RNA, a critical component of RNA granules, can promote or inhibit condensation, alter material properties and substructure organization, and modulate interactions with surfaces. DEAD-box RNA helicases perform ATP-dependent RNA remodeling activities and have been shown to regulate RNA condensates, but the mechanistic details connecting RNA, RNA chaperones, and biological phenotype are largely unexplored. As interest in RNA condensates as an organizing principle in the cell has grown, concerns regarding the evidence supporting their biological significance have emerged. A key challenge for the field is developing technical approaches to measure and manipulate condensates in biological systems and physiologically relevant biochemical reconstitutions. In this proposal, we will investigate the regulation of germ granules, an RNA condensate implicated in germ cell totipotency. Germ granules in C. elegans, have proven a valuable model for understanding RNA condensate properties and biological functions. RNA helicases are critical and conserved regulators of germ granule structure and function in animals including humans. The proposed research aims to establish a framework for understanding the regulation of condensates by RNA helicases and the impact of condensation on RNA helicase activity. To achieve these goals, we will combine analysis of condensates in an animal model with biochemical reconstitution to investigate the molecular mechanics that underpin condensate regulation, dynamics, and function in native cells. We will employ rigorous testing of emerging models through detailed in vitro and quantitative cell-based assays using techniques including genome editing, condensate biophysical measurements, quantitative super-resolution microscopy, and RNA biochemistry. This project uniquely combines our multidisciplinary expertise to unravel fundamental cellular mechanisms and provide new insights that will enable the identification of novel therapeutic targets for condensate-related diseases.